Preparation of injectable bioadhesive hydrogel and application of injectable bioadhesive hydrogel in cartilage regeneration treatment

Abstract

The invention belongs to the technical field of organic-inorganic compounding, and aims to solve the problem of treatment of temporomandibular joint osteoarthritis (TMJOA). Magnesium-gallic acid metal organic framework (Mg-GA MOF) particles are synthesized through a hydrothermal method, hyaluronic acid is subjected to hydroformylation and double-bond modification to obtain OHAMA, and the magnesium-gallic acid metal organic framework (Mg-GA MOF) particles and the OHAMA particles are compounded to prepare the MOF-Gel composite hydrogel. The hydrogel is injectable and high in tissue retention rate, has dual effects of inhibiting pyroptosis and promoting regeneration on condylar cartilage cells, can effectively promote cartilage repair, shows a good effect in TMJOA treatment, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of inorganic and organic composite technology, specifically relating to the preparation of an injectable bioadhesive hydrogel and its application in cartilage regeneration therapy. Background Technology

[0002] Temporomandibular joint osteoarthritis (TMJOA), characterized by pain, clicking, and limited mouth opening, is the most severe subtype of temporomandibular joint disorder. Degenerative changes in the condylar cartilage are one of its main pathological features. Current treatment for TMJOA is primarily symptomatic, and long-term efficacy remains unsatisfactory. The cartilage degeneration in TMJOA manifests as an imbalance between chondrocyte death and regeneration. However, due to the cascade reaction of inflammatory spread during cell death and the limitation of insufficient cartilage regeneration capacity, pathological cartilage degeneration often falls into a vicious cycle, hindering condylar cartilage repair and regeneration. Currently, there is a lack of local treatment strategies to restore cartilage homeostasis. Pyroptosis is a programmed cell death closely related to inflammation and an important form of chondrocyte death. Therefore, designing a biomaterial that both inhibits chondrocyte pyroptosis and promotes cartilage regeneration could potentially improve the therapeutic effect of TMJOA.

[0003] Metal-organic frameworks (MOFs) are compounds formed by the coordination of metal ions and organic ligands. The degradation of the three-dimensional framework structure allows for the controlled release of ions and ligands in a specific ratio. Coordinating two active substances with anti-chondrocyte pyroptosis and chondrocyte regeneration properties to form a bioactive MOF can meet the requirement of simultaneously intervening in cartilage damage and repair. Gallic acid (GA), also known as 3,4,5-trihydroxybenzoic acid, has received widespread attention and recognition for its anti-inflammatory and antioxidant effects. Literature shows that GA inhibits inflammasome activation and pyroptosis. Magnesium ions (Mg...) 2+ Mg is an endogenous trace element. Previous studies have shown that within a certain concentration range, Mg... 2 + It can effectively promote the repair and regeneration of cartilage.

[0004] However, due to the temporomandibular joint's narrow space and high-frequency movement, the direct application of Mg-GA MOF may lead to poor biocompatibility and unstable drug concentrations. Combining it with an injectable delivery system possessing bioadhesive properties is a reasonable solution to this problem.

[0005] Patent CN111214702A discloses an injectable biomimetic repair material for temporomandibular joint disc defects and its preparation method. This repair material consists of two components, precursor solutions A and B, which are mixed and cured under 365nm ultraviolet light to form a gel material for filling the disc defect. However, the preparation process of this material is complex, and it is primarily designed for disc defects rather than condylar cartilage defects, limiting its effectiveness in treating lesions accompanied by cartilage damage. Therefore, developing an injectable biomaterial with good tissue retention and anti-inflammatory / regenerative capabilities is of great significance for the treatment of TMJOA. Summary of the Invention

[0006] To address the imbalance between chondrocyte death and regeneration in TMJOA, GA with anti-pyroptosis effects and Mg that promotes chondrocyte regeneration are used. 2+ We constructed a Mg-GA MOF that improves cartilage homeostasis, loaded it with an injectable bioadhesive scaffold, and injected it into the joint cavity to achieve degenerative cartilage regeneration, treat temporomandibular joint osteoarthritis, and improve patients' quality of life.

[0007] To achieve the above objectives, the present invention provides a method for preparing a bioactive metal-organic framework combined with injectable bioadhesive material by synthesizing metal ions, wherein Mg-GA MOF particles are synthesized by hydrothermal method;

[0008] Hyaluronic acid (HA) was modified in two steps by methacrylation and sodium periodate (NaIO4) oxidation to obtain a photocurable hyaluronic acid hydrogel (OHAMA) with tissue adhesion.

[0009] A homogeneous Mg-GA MOF dispersion obtained by ultrasonic dispersion was combined with an OHAMA colloidal pre-solution, and then crosslinked to obtain a composite hydrogel (MOF-Gel) with a connected macroporous structure.

[0010] Furthermore, the specific steps for the hydrothermal synthesis of Mg-GA MOF particles are as follows: 1g of magnesium chloride (MgCl2) is dissolved in 5mL of deionized water, and 3.8g of GA is dissolved in 45mL of deionized water. GA needs to be heated to aid dissolution (50℃). The MgCl2 solution is added to the GA solution under magnetic stirring at 500rpm and mixed evenly. The pH is adjusted to 8 using 5mol / L KOH solution. After thorough stirring, the solution is transferred to a three-necked flask and heated and stirred in an oil bath at 120℃ for 24h. After the reaction is complete, the precipitate is collected by centrifugation (10000rpm), washed with deionized water, dried, and obtained by sieving to obtain Mg-GA MOF powder.

[0011] Further, the specific steps for the two-step modification of HA by methacrylation and NaIO4 oxidation are as follows: First, prepare HAMA with 80% grafting degree. Take 4g of HA and add it to a three-necked flask. Add 400mL of deionized water and stir mechanically in an ice-water bath at 0℃ until completely dissolved to obtain a 1% HA aqueous solution. Add 8mL of methacrylic anhydride (MA) dropwise through a dropping funnel in an ice-water bath at 0℃, and control the pH of the system to 8.5 with 1M NaOH solution. After the addition is complete, continue stirring in an ice-water bath for 24h. After the reaction is completed, dialyze the solution in deionized water using a 7kDa dialysis bag for 5 days until the conductivity of the dialysate is close to that of deionized water. Change the water 3 times a day during this period. Then, filter the solution to remove the precipitate to obtain HAMA. After freeze-drying, store the HAMA in a -20℃ freezer. Then, oxidize the HAMA to prepare OHAMA. Weigh 2g of HAMA and 4.52g of... NaIO4 was placed in a three-necked flask, and 200 mL of deionized water was added. Under light-protected conditions, the mixture was mechanically stirred (800 rpm) to ensure complete dissolution. After reacting for 2 hours, 4 mL of terminator was added to terminate the reaction. After stirring for another hour, the mixture was transferred to a 3.5 kDa dialysis bag and dialyzed in deionized water for 5 days until the conductivity of the dialysate was close to that of deionized water. The water was changed 3 times a day during this period. The solution was then filtered to remove impurities to obtain OHAMA. OHAMA was then freeze-dried and stored in a -20°C freezer.

[0012] Furthermore, the preparation and injection method of the Mg-GA MOF-loaded OHAMA-gelatin hydrogel (MOF-Gel) is as follows: First, 0.5g of genipin is added to 6mL of glycerol and stirred thoroughly at 60℃ to dissolve the genipin, thus preparing the genipin crosslinking agent; 0.2g of OHAMA and 0.6g of gelatin are dissolved in 10mL of deionized water and then dissolved thoroughly. 0.06g of Mg-GA MOF and 0.01g of photoinitiator LAP are added to the gel solution and dispersed by ultrasound; after the components in the gel solution are fully mixed, 50μL of the genipin crosslinking agent is added to obtain the final mixed gel solution. When preparing hydrogel blocks for cell culture, the mixed gel solution is added to a mold, irradiated with 405nm ultraviolet light for 30s, and then crosslinked in an oven at 37℃ for 12h. After crosslinking is completed, the gel block is removed from the mold, freeze-dried, and then stored. The amount of MOF added can be adjusted according to the actual situation. Adding more MOF can increase the release of the effective components of the composite hydrogel, but the total amount of MOF added should not exceed 20 wt% (W). MOF / W GelOtherwise, excessive local release of MOFs may lead to cytotoxicity. On the other hand, the mechanical properties of the composite hydrogel can be controlled by adjusting the concentration of OHAMA / gelatin and the cross-linking process. Increasing the concentration of OHAMA / gelatin, increasing the amount of genipin, or extending the duration of ultraviolet irradiation can all improve the cross-linking density and mechanical strength of the hydrogel, making it suitable for tissues with different moduli based on different mechanical properties.

[0013] Furthermore, when using the injection in the temporomandibular joint, considering the small space at the temporomandibular joint and the difficulty of UV light penetration, the mixed adhesive is added to the syringe and irradiated with 405nm UV light for 5 seconds. Under the action of the photoinitiator LAP, OHAMA is covalently cross-linked to obtain a pregel with suitable fluidity. This pregel is then injected into the temporomandibular joint area, where it is post-cured through the reaction of genipin and gelatin.

[0014] The invention provides the application of the aforementioned metal ion-synthesized bioactive metal-organic framework combined with injectable bioadhesive materials in temporomandibular joint osteoarthritis to achieve degenerative cartilage regeneration.

[0015] Beneficial effects

[0016] (1) Compared with the prior art, the present invention achieves better tissue adhesion and retention rate by modifying HA, and at the same time ensures good injectability and operability through the double crosslinking of photoinitiator and genipin.

[0017] (2) This invention achieves the synthesis and local administration of Mg-GA MOF, thereby realizing the synthesis and local administration of Mg-GA MOF. 2+ The ratio-controlled release of GA has a dual effect on condylar chondrocytes, inhibiting pyroptosis and promoting regeneration, thus more effectively promoting cartilage tissue repair and regeneration.

[0018] (3) The raw materials for preparing the hydrogel of the present invention are simple and stable, which is conducive to its widespread application. Attached Figure Description

[0019] Figure 1 Scanning electron microscopy (SEM) images of the Mg-GA MOF prepared in Example 1 and Mg 2+ Mapping analyzes photos.

[0020] Figure 2 : A scanning electron microscope image of the cross-sectional structure of the MOF-Gel prepared in Example 1.

[0021] Figure 3 Before photocuring, the MOF-Gel prepared in Example 1 exhibits good flowability and injectability. After double crosslinking with a photoinitiator and genipin, the MOF-Gel becomes morphologically stable.

[0022] Figure 4 The MOF-Gel prepared in Example 1 can produce good bioadhesion in both cartilage and bone tissues, which helps it to remain in the tissue after injection.

[0023] Figure 5 The stress-strain curves of MOF-Gel show that the cross-linked MOF-Gel has good mechanical properties. Figure 6 When the MOF-Gel prepared in Example 1 was co-cultured with primary rat condylar chondrocytes, it inhibited the upregulation of pyroptosis-related proteins NLRP3, GSDMD, and Cleaved CASPASE-1 (Clv.CASP-1) induced by the inflammatory factor TNF-α.

[0024] Figure 7 The MOF-Gel prepared in Example 1 promotes the expression of cartilage differentiation-related marker proteins such as COL II, ACAN, and SOX-9 when co-cultured with rat bone marrow mesenchymal stem cells.

[0025] Figure 8 : This refers to the histological staining of the MOF-Gel prepared in Example 2, which reduced tissue damage and promoted cartilage repair and regeneration after local injection into the joint cavity of TMJOA rats. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. The specific implementation of the invention will be described in detail below with reference to specific embodiments.

[0027] Example 1

[0028] This embodiment provides a method for preparing an injectable bioadhesive hydrogel:

[0029] Synthesis of S1 Mg-GA MOF: 1 g magnesium chloride (MgCl2) was dissolved in 5 mL of deionized water, and 3.8 g gallic acid (GA) was dissolved in 45 mL of deionized water. The MgCl2 solution was added to the GA solution under magnetic stirring at 500 rpm and mixed thoroughly. The pH was adjusted to 8 with 5 mol / L KOH solution, and the mixture was transferred to a three-necked flask. The mixture was heated in an oil bath with stirring (120℃, 300 rpm) for 24 h. After the reaction was complete, the precipitate was collected by centrifugation (10000 rpm), washed with deionized water, dried, and sieved to obtain Mg-GA MOF powder.

[0030] Synthesis of S2 OHAMA: First, prepare HAMA with 80% grafting degree. Add 4g of HA to a three-necked flask, add 400mL of deionized water, and mechanically stir (800rpm) in an ice-water bath at 0℃ until completely dissolved to obtain a 1% HA aqueous solution. Add 8mL of MA dropwise in an ice-water bath at 0℃, controlling the pH of the system to 8.5 with 1M NaOH solution. After the addition is complete, continue stirring in an ice-water bath for 24h. After the reaction is complete, dialyze the solution in deionized water using a 7kDa dialysis bag for 5 days, changing the water 3 times a day. Filter to remove the precipitate to obtain HAMA, freeze-dry and store at -20℃. Next, HAMA was oxidized to prepare OHAMA. 2g of HAMA and 4.52g of sodium periodate (NaIO4) were weighed and placed in a three-necked flask. 200mL of deionized water was added, and the mixture was mechanically stirred (800rpm) in the dark to ensure complete dissolution. After reacting for 2 hours, 4mL of ethylene glycol was added as a terminator to terminate the reaction. After stirring for another hour, the mixture was transferred to a 3.5kDa dialysis bag and dialyzed in deionized water for 5 days, with the water changed 3 times a day. Impurities were removed by filtration to obtain OHAMA, which was then freeze-dried and stored at -20℃.

[0031] Preparation of S3 MOF-Gel hydrogel: 0.5g genipin was added to 6mL glycerol and stirred thoroughly at 60℃ to dissolve the genipin, thus preparing the genipin crosslinking agent. 0.2g OHAMA and 0.6g gelatin were dissolved in 10mL deionized water and then dissolved thoroughly. 0.06g Mg-GA MOF and 0.01g photoinitiator LAP were added and ultrasonically dispersed. After all components were thoroughly mixed, 50μL of the genipin crosslinking agent solution was added to obtain the final mixed gel. S4 The mixed gel was injected into a syringe or molding die, irradiated with 405nm UV light for 30s, crosslinked in a 37℃ oven for 12h, and then freeze-dried for storage.

[0032] Experimental detection of pyroptosis-related protein expression levels: Primary condylar chondrocytes from 3-week-old female rats were cultured. Stimulation with the inflammatory factor TNF-α significantly increased the expression of pyroptosis-related proteins such as NLRP3, GSDMD, and CASPASE-1. However, co-culturing with MOF-Gel during TNF-α stimulation resulted in a certain decrease in the expression levels of these pyroptosis-related proteins, suggesting that MOF-Gel can alleviate chondrocyte pyroptosis. (Appendix) Figure 6 )

[0033] Detection of chondrogenic differentiation-related protein expression levels: Primary bone marrow mesenchymal stem cells (MMSCs) were extracted from 3-week-old female rats and cultured for chondrogenic differentiation. The results showed that after co-culturing with MOF-Gel, chondrogenic differentiation marker proteins such as COL II, ACAN, and SOX-9 in the MMSCs were significantly upregulated, suggesting that MOF-Gel promotes chondrogenic regeneration. (Appendix) Figure 7 )

[0034] Example 2

[0035] This embodiment provides an application of injectable bioadhesive hydrogel in the treatment of temporomandibular joint osteoarthritis:

[0036] The preparation of S1-S3 is the same as in Example 1.

[0037] S4 Take fresh mixed adhesive solution and add it to the syringe. First, irradiate it with 405nm ultraviolet light for 5s to form a pregel solution with a certain fluidity. Then, immediately inject it into the temporomandibular joint of the rat. The injected adhesive solution will be post-cured through the reaction of genipin and gelatin.

[0038] Comparative Example 1

[0039] This comparative example provides a method for preparing an injectable bioadhesive hydrogel. The preparation method is the same as in Example 2, except that the hydrogel prepared in this comparative example does not contain Mg-GA MOF: 0.2 g of OHAMA and 0.6 g of gelatin are dissolved in 10 mL of deionized water and fully dissolved. 0.01 g of photoinitiator LAP is added and ultrasonically dispersed. After the components are fully mixed, 50 μL of genipin crosslinking agent is added to obtain a mixed gel solution. The mixed gel solution is injected into a syringe or molding die, irradiated with 405 nm ultraviolet light for 30 s, crosslinked in an oven at 37 °C for 12 h, and then freeze-dried for storage.

[0040] Example 2 vs. Comparative Example 1: An in vivo experiment was conducted using a rat model of temporomandibular joint osteoarthritis (OA). Sixteen 8-week-old female SD rats were injected bilaterally into their temporomandibular joints with 0.5 mg / 50 μL sodium iodoacetate (MIA) to induce the OA model. One week later, they were randomly divided into two groups. The first group received the hydrogel matrix described in Comparative Example 1 (labeled as Gel) in the left temporomandibular joint, while the second group received the optimized composite hydrogel described in Example 2 (labeled as MOF-Gel) in the left temporomandibular joint. All rats received an equal volume of saline in their right temporomandibular joints as a self-control. Two and four weeks after treatment, samples were collected from four rats in each group to evaluate the histological morphology of the joint tissue sections. The results showed that significant degenerative changes occurred in the condylar cartilage after OA modeling. Injection of the Gel hydrogel described in Comparative Example 1 somewhat alleviated the degree of cartilage lesions, but significant cartilage degeneration remained. Injection of the MOF-Gel described in Example 2 significantly improved the cartilage morphology, making it more uniform, intact, and close to the morphology of normal condylar cartilage. (Appendix) Figure 8 ).

Claims

1. A method for preparing an injectable bioadhesive hydrogel, characterized in that, Includes the following steps: Mg-GA MOF particles were synthesized via a hydrothermal method. Hyaluronic acid (HA) was modified in two steps by methacrylation and sodium periodate oxidation to obtain a photocurable hyaluronic acid hydrogel (OHAMA) with tissue adhesion ability. A homogeneous Mg-GA MOF dispersion obtained by ultrasonic dispersion was combined with an OHAMA colloidal pre-solution, and then crosslinked to obtain a composite hydrogel (MOF-Gel) with a connected macroporous structure.

2. The hydrothermal synthesis of Mg-GA MOF particles according to claim 1, characterized in that: Magnesium chloride (MgCl2) and gallic acid (GA) were dissolved separately in deionized water. The MgCl2 solution was added to the GA solution under magnetic stirring and mixed evenly. The pH was adjusted to 8 using KOH solution. After thorough stirring, the solution was transferred to a three-necked flask, heated and stirred in an oil bath. After the reaction was completed, the precipitate was collected by centrifugation, washed with deionized water, dried, and obtained Mg-GA MOF powder by sieving.

3. The preparation method according to claim 1, which modifies HA in two steps by methacrylation and sodium periodate (NaIO4) oxidation, is characterized in that: First, HAMA with 80% methacrylamide grafting degree was prepared. HA was added to deionized water and mechanically stirred in an ice-water bath at 0°C until completely dissolved to obtain a 1% HA aqueous solution. 8 mL of methacrylic anhydride (MA) was added dropwise through a dropping funnel in an ice-water bath at 0°C, with the pH of the system controlled at 8.5 using NaOH solution. After the addition was complete, stirring was continued in an ice-water bath. After the reaction was completed, the solution was dialyzed and then filtered to remove the precipitate to obtain HAMA.

4. The preparation method according to claim 3, wherein HAMA is prepared by oxidation treatment, characterized in that: HAMA and NaIO4 were placed in a three-necked flask, and deionized water was added. Under light-protected conditions, the mixture was mechanically stirred until it was fully dissolved. Then, a terminator was added to terminate the reaction. After stirring, the mixture was transferred to a dialysis bag and dialyzed in deionized water until the conductivity of the dialysate was close to that of deionized water. Finally, the solution was filtered to remove impurities and obtain OHAMA.

5. The preparation method according to claim 1, for the preparation and injection of OHAMA-gelatin hydrogel (MOF-Gel) loaded with Mg-GA MOF, characterized in that: Genipin was first added to glycerol and stirred thoroughly at 60°C to dissolve it, thus preparing the genipin crosslinking agent. OHAMA and gelatin were dissolved in deionized water and then Mg-GAMOF and the photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) were added to the gel solution and dispersed by ultrasound. After the components in the gel solution were fully mixed, the genipin crosslinking agent was added to obtain a mixed gel solution. When preparing hydrogel blocks for cell culture, the mixed gel solution was added to a mold, irradiated with 405nm ultraviolet light for 30s, and then crosslinked in an oven at 37°C. After crosslinking was completed, the gel block was removed from the mold, freeze-dried, and stored.

6. The preparation method according to claim 1, wherein the hydrogel material is applied to various bone and joint injections in vivo, characterized in that: The freshly mixed adhesive solution is added to a syringe and irradiated with 405nm ultraviolet light for 5 seconds. Under the action of the photoinitiator LAP, OHAMA is covalently cross-linked to obtain a pregel solution with suitable flowability. After injection into the target site, post-curing is achieved through the reaction of genipin and gelatin. Good mechanical properties are obtained while ensuring injection flowability.

7. A composite hydrogel material for TMJOA and other cartilage regeneration therapies, characterized in that: The MOF-Gel composite hydrogel material for cartilage regeneration therapy is prepared according to any one of claims 1-6.

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